ARTICLE pubs.acs.org/jmc
Antitumor Agents 288: Design, Synthesis, SAR, and Biological Studies of Novel Heteroatom-Incorporated Antofine and Cryptopleurine Analogues as Potent and Selective Antitumor Agents Xiaoming Yang,† Qian Shi,*,† Shuenn-Chen Yang,§ Chi-Yuan Chen,§ Sung-Liang Yu,||,3 Kenneth F. Bastow,‡ Susan L. Morris-Natschke,† Pei-Chi Wu,‡ Chin-Yu Lai,^ Tian-Shung Wu,O Shiow-Lin Pan,^ Che-Ming Teng,^ Jau-Chen Lin,§ Pan-Chyr Yang,*,^,§,# and Kuo-Hsiung Lee*,†,O Natural Products Research Laboratories and ‡Division of Medicinal Chemistry and Natural Products, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599-7568, United States § Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan Department of Clinical Laboratory Sciences and Medical Biotechnology, ^Department of Internal Medicine, College of Medicine, and # Division of Genomic Medicine, Research Center for Medical Excellence, National Taiwan University, Taipei, Taiwan 3 Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan O Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung 404, Taiwan
)
†
bS Supporting Information ABSTRACT: Novel heteroatom-incorporated antofine and cryptopleurine analogues were designed, synthesized, and tested against a panel of five cancer cell lines. Two new S-13-oxo analogues (11 and 16) exhibited potent cell growth inhibition in vitro (GI50: 9 nM and 20 nM). Interestingly, both compounds displayed improved selectivity among different cancer cell lines, in contrast to the natural products antofine and cryptopleurine. Mechanism of action (MOA) studies suggested that R-antofine promotes dysregulation of DNA replication during early S phase, while no similar effects were observed for 11 and 15 on corresponding replication initiation complexes. Compound 11 also showed greatly reduced cytotoxicity against normal cells and moderate antitumor activity against HT-29 human colorectal adenocarcinoma xenograft in mice without overt toxicity.
’ INTRODUCTION Phenanthroindolizidines and phenanthroquinolizidines, which are natural alkaloids isolated from several plant species such as the Asclepiadacae and Moracae family,1 have been investigated intensively in recent years, due to their interesting biological characteristics, including antimicrobial, antiangiogenic, and anti-inflammatory effects,28 as well as significant cytotoxicity as demonstrated in the NCI 60 cell-line assay (Figure 1).9 Further studies have shown that these natural products exhibit not only strong inhibitory activity against cancer cell growth but also significant effects on cancer cells resistant or cross-resistant to many anticancer drugs in the market.10 Thus, this important class of chemical entities may potentially augment our present arsenal of anticancer drugs.11,12 Although the biological target(s) and MOA of these natural products are currently still unclear, some interesting findings have been reported. A possible mechanism of action might be inhibition of NF-kB signaling, a well-known pathway in the antiapoptosis and survival of cancer cells, as well as regulation of P-glycoprotein.13 Other hypotheses, such as inhibition of protein synthesis during the chain elongation stage,14 targeting ribosomal subunits (low-affinity binding pockets have been identified in the 40S, 60S, 70S, and 80S subunits),1517 inhibition of r 2011 American Chemical Society
Figure 1. Representative structures of phenanthroindolizidines and phenanthroquinolizidines.
hypoxia-inducible factor 1 (HIF-1),18 inhibition of thymidylate synthase (TS) and dihydrofolate reductase (DHFR),1921 suppression of activator protein-1 (AP-1) and the cAMP response element (CRE) signaling pathway, reduction of cell cycle regulatory proteins such as cyclin D1, cyclin B1, CDK1, CDK2, and CDK4, etc.10 have also been reported. In addition, evidence has suggested that certain structural analogues might not be functional analogues,22 and thus, multiple biological targets might exist. Received: March 22, 2011 Published: June 13, 2011 5097
dx.doi.org/10.1021/jm200330s | J. Med. Chem. 2011, 54, 5097–5107
Journal of Medicinal Chemistry
ARTICLE
Scheme 1a
a
Reagents and conditions: (a) (Boc)2O, Et3N, CH2Cl2; (b) (i) Py 3 SO3, DMSO, Et3N, CH2Cl2; (ii) RNH2, HOAc, NaBH3CN, MeOH; (iii) TFA, CH2Cl2; (iv) K2CO3, MgSO4, HCHO, CH2Cl2.
Despite their promise, the potential development of antofine, cryptopleurine, or related natural or synthetic alkaloids as promising drug candidates has been limited, to a large degree, by severe CNS toxicity, such as disorientation and ataxia,23 and low natural availability. All of these factors combine to justify an urgent need for improving the pharmacokinetic and pharmacodynamic properties, such as polarity, of this compound class through rational structural modifications. However, only limited studies have been reported in this regard, e.g., introduction of a hydroxy group at C14 of phenanthroindolizidines,24 construction of phenanthrene-based tylophorine analogues,2527 and N atom incorporation at C13a of tylophorine,28 probably due to the lack of a convergent and efficient synthetic methodology. Recently, our group reported a new strategy suitable for producing numerous new phenanthroindolizidine and phenanthroquinolizidine analogues with E-ring modifications.29 The versatility and significance of this approach lie in the fact that it not only facilitates the SAR study of E-ring variations but also, even more importantly, generates a group of analogues carrying a heterocyclic E-ring or other polar moieties to potentially overcome CNS toxicity. CNS toxicity is closely associated with the blood brain barrier (BBB) penetration of a molecule, which is in large part related to its physicochemical properties, such as lipophilicity (cLogP) or polarity, molecular size and charge, polar surface area (PSA), or hydrogen bonding potential, etc., although other factors may also play a role.3032 Herein, we report the design, synthesis, in vitro anticancer activity, SAR, and mechanistic studies of new antofine and cryptopleurine derivatives with a N or O atom incorporated in the E-ring. The in vivo antitumor activity for the most active compound (11) is also reported.
’ RESULTS AND DISCUSSION Initially, antofine analogues bearing a N atom at position C12 were designed and synthesized. The key intermediate 1 was prepared via a procedure recently reported by our group.27 The amino group of 1 (S or R isomer) was protected initially with a Boc group to give 2. Then the hydroxy group was oxidized with Py 3 SO3 to an aldehyde, which was then converted to various secondary amines through reductive amination using NaBH3CN. After the removal of the Boc group, 3a3l were obtained through cyclization with formaldehyde (Scheme 1). Compounds 3a3l were then screened against four cancer cell lines, A549 (lung), DU-145 (prostate), KB (nasopharyngeal), and HCT-8 (colon). The screening results are shown in Table 1. In comparison with R-antofine, all 12 compounds exhibited substantially decreased activity with an average GI50 over 1 μM. In addition, no cell-line selectivity was observed. Compound 3f (R = Ph) was the least potent (inactive) with an average GI50 value
greater than 20 μM. Insertion of a single methylene group between the N and phenyl ring (3a and 3j, R = CH2Ph) resulted in greater activity (with the R isomer approximately 2-fold better than the S isomer), while addition of a second methylene group (3d, R = CH2CH2Ph) did not improve activity any further. Generally, the compounds with an aliphatic amino moiety [cyclic (3g), straight chain (3c, 3 h, and 3l), or branched chain (3b)] showed slightly better activity than those bearing an aromatic moiety. Compounds 3h and 3l (R = Me) showed the greatest potency among all compounds, and the R isomer (3l) was slightly more potent than its S enantiomer (3h). Conversely, 3i (S isomer) was 2-fold more active than its R isomer (3k). These data implied that introduction of a N atom at position C12 of antofine might not improve the cytotoxicity against cancer cell lines, even though it did increase the polarity as predicted by PreADMET.33 Next, we studied the effect of N-incorporation in cryptopleurine, both at positions C12 and C13. For the latter compound series, 2 (S or R) was converted to 4 through oxidation with Py 3 SO3 and subsequent reductive amination using glycine methyl ester hydrochloride. The E ring was formed by sequential deprotection with HCl/MeOH and cyclization with Et3N/MeOH. The resulting intermediate was further protected with a Boc group to give 5 for easy purification. The lactam carbonyl was then reduced to a methylene using BMS/THF to afford 6. After the removal of the Boc group, 13-aza-cryptopleurines (7aq) were obtained through either acylation or reductive amination (Scheme 2). The GI50 values of 7aq are listed in Table 2. Although the 13-aza analogues showed significantly reduced activity as compared with that of R-cryptopleurine, some interesting results were observed. Compound 7g, with a N-cyclopropylmethyl substituent, was the most potent analogue against the HCT-8 cell line with a GI50 value of 0.25 μM, in addition to better selectivity against A549 and HCT-8 versus DU145 and KB. However, 7h, with a N-cyclopropyl substituent, one CH2 shorter than 7g, showed dramatically reduced activity. Similar results were observed from the comparison of 7c and 7f, as well as antofine analogues 3f, 3a, and 3d, suggesting that the length of the side chain on nitrogen affects the anticancer activity. It is also interesting to note that 7g was much more potent than its R isomer 7q, whereas 7m and 7p exhibited the reversed order of activity, although to a less significant degree. Of all the tested analogues in this series, compound 7a, the hydrochloride salt of 13-aza-cryptopleurine, showed considerable anticancer activity against all four tested cell lines, indicating that 7a might be a promising lead meriting further investigation. In the following studies, a series of cryptopleurine analogues (10aj) with N replacement at position C12 were synthesized to 5098
dx.doi.org/10.1021/jm200330s |J. Med. Chem. 2011, 54, 5097–5107
Journal of Medicinal Chemistry
ARTICLE
Table 1. GI50 Values of 12-Aza-antofines 3al against Four Cancer Cell Lines
compd.
R
Config.
A549 (μM)
DU145 (μM)
KB (μM)
HCT-8 (μM)
3a
-Bn
S
5.30 ( 1.02
4.95 ( 1.24
5.70 ( 1.52
3.63 ( 0.81
3b
-iBu
S
2.00 ( 0.36
6.60 ( 1.45
4.40 ( 1.23
1.82 ( 0.45
3c
-nPr
S
3.94 ( 0.78
7.38 ( 1.55
6.64 ( 1.73
2.71 ( 0.49
3d
-(CH2)2Ph
S
6.62 ( 2.04
10.20 ( 3.13
10.03 ( 2.11
7.47 ( 1.68
3e
20 OH-Et
S
4.41 ( 0.97
6.36 ( 1.29
7.83 ( 1.59
10.77 ( 2.44
3f
-Ph
S
>20
>20
>20
>20
3g 3h
-cPr -Me
S S
1.80 ( 0.36 0.66 ( 0.11
3.70 ( 0.81 2.03 ( 0.38
2.71 ( 0.89 1.72 ( 0.55
1.85 ( 0.48 1.00 ( 0.19
3i
-NMe2
S
1.67 ( 0.37
2.26 ( 0.47
2.31 ( 0.65
2.01 ( 0.54
3j
-Bn
R
2.68 ( 0.51
4.62 ( 1.16
3.41 ( 0.78
1.87 ( 0.43 (KBvin)
3k
-NMe2
R
4.15 ( 0.87
6.01 ( 1.50
2.38 ( 0.60
10.98 ( 2.92 (KBvin)
3l
-Me
R
0.66 ( 0.10
1.00 ( 0.23
0.79 ( 0.21
0.66 ( 0.16
R
22 ( 7 nM
25 ( 5 nM
36 ( 8 nM
-
R-antofine
Scheme 2a
a Reagents and conditions: (a) (i) Py 3 SO3, DMSO, Et3N, CH2Cl2; (ii) glycine methyl ester hydrochloride, Et3N, HOAc, NaBH3CN, MeOH; (b) (i) HCl, MeOH; (ii) MeOH, Et3N; (iii) (Boc)2O, Et3N, CH2Cl2; (c) BMS, THF; (d) (i) TFA, CH2Cl2; (ii) RCOCl, Et3N, CH2Cl2 or RCHO, Et3N, HOAc, NaBH3CN, MeOH.
explore their anticancer activity. Compound 2 was converted to vinylmethyl ether 8 in two steps, oxidation followed by a Wittig reaction using Ph3P=CH2OMe. Compound 8 was then hydrolyzed with Hg(OAc)2 to give aldehyde 9. The targets, 12-azacryptopleurines 10aj, were prepared using a strategy similar to that described in the synthesis of compounds 3al (Scheme 3). The GI50 values for 10aj are listed in Table 3. Overall, the results suggested that N-incorporation at position C12 of cryptopleurine diminished the anticancer activity (lowest GI50 value was 1.12 μM for a 12-aza analogue 10j versus nM values for cryptopleurine). Anticancer activity patterns similar to those described above with the other compound series were observed.
Compounds with bulky or aromatic groups, such as a phenyl or benzyl moiety (10b or 10d), exhibited lower activity (>20 μM), as also observed in the above two series of analogues. Introducing a polar group, such as hydroxyethyl in 10a/10i and N,Ndimethylamino in 10f/10h, did not lead to increased activity, although some moderate cell-line selectivity was found. The stereochemistry of the analogues also affected the anticancer activity (compare 10a/10i, 10c/10g, and 10f/10h), as was also observed in the above two analogue series, although the trends were neither significant nor consistent. Interestingly, in the comparison of 10j and 7q (N12 vs N13 substitution), the former compound was significantly more potent than the latter. 5099
dx.doi.org/10.1021/jm200330s |J. Med. Chem. 2011, 54, 5097–5107
Journal of Medicinal Chemistry
ARTICLE
Table 2. GI50 Values of 13-Aza-cryptopleurines 7aq against Four Cancer Cell Lines
compd.
R
config.
A549 (μM)
DU145 (μM)
KB (μM)
HCT-8 (μM)
7a
H, HCl salt
S
1.73 ( 0.52
2.08 ( 0.27
1.79 ( 0.37
1.62 ( 0.56
7b
-Et
S
7.13 ( 3.86
10.48 ( 2.51
8.24 ( 1.89
9.45 ( 3.21
7c
-CO2Me
S
10.93 ( 5.10
12.19 ( 3.04
11.32 ( 2.25
12.05 ( 2.58
7d
-Ac
S
11.65 ( 1.88
11.94 ( 2.22
15.46 ( 2.78
13.82 ( 2.80
7e
-Ms
S
14.52 ( 3.02
16.97 ( 3.14
17.52 ( 3.10
17.83 ( 3.63
7f
-CH2CO2Me
S
14.74 ( 2.49
>20
14.65 ( 2.88
14.03 ( 2.12
7g 7h
-CH2cPr -cPr
S S
0.79 ( 0.11 >20
2.94 ( 0.45 17.92 ( 5.28
6.57 ( 1.56 >20
0.25 ( 0.11 13.02 ( 3.03
7i
-Bz
S
15.00 ( 3.77
15.71 ( 4.32
>20
18.11 ( 4.91
7j
20 OH-Et
S
14.08 ( 5.49
12.62 ( 5.61
8.62 ( 2.44
7.41 ( 1.30
7k
-PO(OMe)2
S
12.18 ( 3.18
14.66 ( 4.59
8.09 ( 1.02
10.42 ( 2.21 (KBvin)
7l
-Me
S
9.73 ( 1.81
7.13 ( 1.05
5.30 ( 0.46
6.45 ( 1.03
7m
-Bn
S
>20
>20
16.85 ( 5.64
15.24 ( 3.35 (KBvin)
7n
-CONMe2
R
14.08 ( 1.86
13.12 ( 1.98
12.32 ( 2.15
11.41 ( 2.10 (KBvin)
7o 7p
-iBu -Bz
R R
12.86 ( 2.31 16.43 ( 6.08
12.24 ( 4.04 11.87 ( 3.92
11.25 ( 2.36 12.14 ( 2.91
9.96 ( 1.89 (KBvin) 7.64 ( 1.07 (KBvin)
7q
-CH2cPr
R
>20
>20
>20
13.44 ( 2.42 (KBvin)
R
1.38 ( 0.56 nM
1.59 ( 0.53 nM
1.51 ( 0.33 nM
1.09 ( 0.20 nM
crypto-pleurine
Scheme 3a
a
Reagents and conditions: (a) (i) Py 3 SO3, DMSO, Et3N, CH2Cl2; (ii) Ph3P+CH2OMeCl, THF, KOtBu; (b) Hg(OAc)2, THF, H2O; (c) (i) RNH2, HOAc, NaBH3CN, MeOH; (ii) TFA, CH2Cl2; (iii) K2CO3, MgSO4, HCHO, CH2Cl2.
Likewise, 10g was more potent than 7o, likely indicating that the relocation of the N atom could cause a possible conformational change, which led to a differentiation in biological activity. Even though the above modifications of antofine and cryptopleurine by N incorporation in the E-ring did not provide analogues with enhanced or at least comparable anticancer activity to the natural alkaloids, we extended our studies by
investigating the effect of O atom incorporation at corresponding positions. Unfortunately, introduction of the oxygen atom at position C12 of antofine was not successful due to the instability of the resulting analogue. However, incorporation of the O atom into the E-ring of cryptopleurine at position C12 or C13 was achieved, as described in Scheme 4. Reaction of 1 with chloroacetyl chloride furnished the intermediate amide, which underwent intramolecular nucleophilic attack using NaH/THF and subsequent reduction using BMS/ THF to afford S-13-oxo-cryptopleurine 11 and its R isomer 12. For the O replacement at position C12 of cryptopleurine, the aldehyde of 9 was first converted to an hydroxymethyl group by NaBH4 reduction, followed by removal of Boc and subsequent cyclization using HCHO to give S-12-oxo-cryptopleurine 13 and its R isomer 14 (Scheme 4). A cytotoxicity evaluation of 1114 against four cancer cell lines was conducted, and the results are summarized in Table 4. It is exciting to note that 11, with O at position C13, exhibited potent nanomolar anticancer activity with greater selectivity toward the HCT-8 tumor cell line (GI50 = 9 nM). In contrast, compound 12, the R-enantiomer of 11, showed significantly decreased activity. As for the incorporation of O at position C12, both analogues 13 and 14 exhibited much lower potency than Rcrytopleurine with GI50 >1 μM. The slight structural variation among 1114 resulted in significantly different biological activity. Compound 11 might achieve a specific structural conformation that is required for interacting with the target protein leading to the observed anticancer activity. However, further studies are 5100
dx.doi.org/10.1021/jm200330s |J. Med. Chem. 2011, 54, 5097–5107
Journal of Medicinal Chemistry
ARTICLE
Table 3. GI50 Values of Analogues 10aj against Four Cancer Cell Lines
compd.
R
config.
A549 (μM)
DU145 (μM)
KB (μM)
HCT-8 (μM)
10a
-20 -OH-Et
S
5.68 ( 1.36
1.99 ( 0.52
1.59 ( 0.36
10b
-Ph
S
>20
>20
>20
>20
10c
-iBu
S
1.52 ( 0.38
2.30 ( 0.55
5.06 ( 1.82
1.36 ( 0.29
10d
-Bn
S
>20
>20
>20
>20
10e
-cPr
S
4.30 ( 0.77
14.33 ( 3.80
11.47 ( 3.72
5.73 ( 1.14
10.65 ( 3.73
10f
-NMe2
S
1.78 ( 0.43
2.97 ( 0.51
3.08 ( 0.50
2.37 ( 0.78
10g 10h
-iBu -NMe2
R R
5.11 ( 1.62 5.20 ( 1.02
1.66 ( 0.43 3.00 ( 0.65
1.45 ( 0.22 2.84 ( 0.56
7.32 ( 1.90 (KBvin) 5.30 ( 1.18 (KBvin)
10i
-20 -OH-Et
R
2.48 ( 0.36
1.46 ( 0.33
2.22 ( 0.41
>20 (KBvin)
10j
-CH2cPr
R
1.12 ( 0.29
1.20 ( 0.28
2.10 ( 0.39
5.68 ( 1.53 (KBvin)
R
1.38 ( 0.56 nM
1.59 ( 0.53 nM
1.51 ( 0.33 nM
1.09 ( 0.20 nM
crypto-pleurine
Scheme 4a
a Reagents and conditions: (a) (i) ClCH2COCl, Et3N, CH2Cl2; (ii) NaH, THF, reflux; (iii) BMS, THF; (b) (i) NaBH4, MeOH; (ii) TFA, CH2Cl2; (iii) K2CO3, MgSO4, HCHO, CH2Cl2.
needed to verify such a hypothesis, as the decreased activity of 13 and 14 could also possibly be due to molecular instability (the hemiaminal ether E-ring). Our study of O incorporation in antofine/cryptopleurine-type compounds was also extended to the previously reported 7-membered E-ring analogue 15.29 Its O-containing analogue 16 was synthesized using procedures similar to those for 11 (Scheme 5). First, the aldehyde of 9 was reduced to a hydroxymethyl group followed by the removal of Boc. The resulting amino intermediate was then reacted with chloroacetyl chloride at 0 C to provide the chloroacetamide, which was subjected to ring closure and subsequent reduction of the amide carbonyl to a methylene with BMS to give compound 16 in 24% yield over five steps. When tested in parallel for cytotoxicity, both 15 and 16 were more active against A549 and HCT-8 cancer cell lines than
against DU145 and KB (Table 5). Compound 16 was slightly more active than 15 against DU145 and KB cell proliferation, while it was slightly less active against A549 and HCT-8. These results suggested that the remarkable cell-line selectivity of 15 was somewhat decreased by introducing an oxygen atom at C13. Nevertheless, both analogues exhibited strong potency against the HCT-8 cell line, which indicated that phenanthroindolizidine and phenanthroquinolizidine derivatives with a 7-membered E-ring have a new and interesting structural scaffold and could have a great potential for further development as selective anticolorectal cancer agents. In consideration of the impressive cytotoxicity, we next aimed our study at the potential mechanism(s) of action of the representative analogues 11 and 15, as well as R-antofine. Our preliminary cDNA microarray data indicated that R-antofine had a profound influence on the major replication initiation complexes ORC (origin recognition complex) and MCM (DNA replication licensing factor) in early S phase. The DNA replication licensing factors Cdt1 (DNA replication factor) and CDC6 (cell division control protein 6) were also up-regulated in a dose- or time-dependent manner when CL15 cells were exposed to R-antofine for 24 and 48 h, as confirmed by RT-PCR (0.01 μg/mL, Figure 2) and Western blot analysis (Figure 3). These data indicated that R-antofine promotes dysregulation of DNA replication in lung cancer cells. However, it is not clear whether the cytotoxic effect of R-antofine is directly correlated with promoting dysregulation of DNA replication. In general, the effects of 11 and 15 were not significant, falling in between those produced by R-antofine and control. An in-depth investigation on diverse cell events should be helpful for further clarification of their mechanism of action. Obviously, the difference in the E-ring structure is involved in these observed effects, and the E-ring is not totally indispensable. Compound 11 was then selected for an in vivo study in mice against HT-29 human colorectal adenocarcinoma xenograft. Mice in group 1 received the vehicle and served as the control for all treatment groups. The median time to end point (TTE) for mice in the control group was 16.3 days with a range of 5101
dx.doi.org/10.1021/jm200330s |J. Med. Chem. 2011, 54, 5097–5107
Journal of Medicinal Chemistry
ARTICLE
Table 4. GI50 Values of Analogues 1114 against Four Cancer Cell Lines
compd.
config.
A549
DU145
KB
HCT-8
11
S
23 ( 5 nM
67 ( 10 nM
37 ( 9 nM
9 ( 5 nM
12 13
R S
13.95 ( 4.74 μM 1.79 ( 0.27 μM
7.05 ( 1.25 μM 1.82 ( 0.31 μM
6.68 ( 1.52 μM 1.66 ( 0.33 μM
3.45 ( 0.56 μM 1.45 ( 0.24 μM
14
R
1.37 ( 0.29 μM
1.29 ( 0.36 μM
1.10 ( 0.28 μM
1.26 ( 0.20 μM
crypto-pleurine
R
1.38 ( 0.56 nM
1.59 ( 0.53 nM
1.51 ( 0.33 nM
1.09 ( 0.20 nM
Scheme 5a
Reagents and conditions: (a) (i) NaBH4, MeOH; (ii) TFA, CH2Cl2; (iii) ClCH2COCl, Et3N, CH2Cl2, 0 C; (iv) NaH, THF, reflux; (v) BMS, THF, 24% for 5 steps. a
Table 5. GI50 Values of Compounds 15 and 16 compd. config. A549 (nM) DU145 (nM) KB (nM) HCT-8 (nM) 15
R
25 ( 5
179 ( 36
102 ( 19
10 ( 3
16
S
41 ( 9
119 ( 33
68 ( 10
20 ( 6
13.821.9 days. Compound 11, i.v. at 20 mg/kg (qd to end), produced a median TTE of 21.3 days and caused 1.1% maximum group body weight loss on day 12, indicating statistically significant antitumor activity (P < 0.05) and no obvious toxicity. When dosed i.v./i.p. at 20 mg/kg (bid to end), 11 produced a median TTE of 20.9 days and caused 7.2% maximum group body weight loss occurring on day 19. One tumor remained in the study on day 29, and one treatment-related (TR) death occurred on day 20 (Figures 4 and 5). When dosing frequency was increased, no improved efficacy was observed. Interestingly, a similar phenomenon was also reported in previous studies. For instance, the effectiveness of phenanthroindolizidine analogues with C14-OH was found to be highly schedule dependent, e.g., the therapeutic index of DCB-3503 may be lost if given daily instead of every three days.34 Therefore, a more rationally designed dosing regimen is being investigated in order for the compounds to exert their best tumor inhibitory effects and meanwhile reduce toxicity that could result from unnecessary repetitive dosing. Moreover, we tested compound 11 against human umbilical vein endothelial cells (HUVECs, normal cells), and the results indicated that the cytotoxicity of
R-cryptopleurine was reduced by 30-fold through our modification, as shown in Figure 6 (GI50 320 nM vs 11 nM). Such improved selectivity against cancer cells over normal cells demonstrates that our newly synthesized analogues can potentially reduce unwanted side effects in vivo. In summary, compound 11 exhibited potent in vitro anticancer activity with improved cell line selectivity and moderate antitumor activity against HT-29 human colorectal adenocarcinoma in male nude-athymic mice at a dose of 20 mg/kg without causing obvious toxicity.
’ CONCLUSIONS New synthetic antofine and cryptopleurine analogues with varied E-ring size or heteroatom incorporation were designed and prepared. Two analogues, 11 and 16, were the most active compounds against tested cancer cell lines, with interestingly improved selectivity against HCT-8 cell growth. Mechanistic studies indicated that R-antofine is capable of promoting dysregulation of DNA replication in early S phase, whereas no similar results were found for compounds 11 and 15. It is still unclear as to how this activity correlates with the potent cytotoxicity of antofine, and further investigation is ongoing. In our in vivo antitumor study, we found that 11 had improved activity on HUVECs and moderate antitumor effect on human HT-29 adenocarcinoma xenograft in mice. However, further studies in other tumor models are warranted to find out which type of tumor affords the most sensitivity and what treatment protocol 5102
dx.doi.org/10.1021/jm200330s |J. Med. Chem. 2011, 54, 5097–5107
Journal of Medicinal Chemistry provides the best therapeutic index. Moreover, compound 16 appears to be an interesting analogue that necessitates in-depth investigation, and results will be reported in due course. Additionally, CNS toxicity will also be explored in the future.
’ EXPERIMENTAL SECTION All chemicals were used as purchased. Melting points were measured using a Fisher Johns melting apparatus without correction. Proton nuclear magnetic resonance (1H NMR) spectra were measured on a
ARTICLE
300 MHz Gemini or a Varian Inova (400 MHz) NMR spectrometer with TMS as the internal standard. The solvent used was CDCl3 unless otherwise indicated. Mass spectra were recorded on a Shimazu-2010 LC/MS/MS instrument equipped with a TurboIonsSpray ion source. All final target compounds were characterized and determined as at least >95% pure by analytical HPLC.
(S)-N-Boc-(6,7,10-trimethoxy-1,2,3,4-tetrahydrodibenzo[f,h]isoquinolin-3-yl)methanol (2). (Boc)2O (3.14 g, 14.40 mmol) was added to the amino-alcohol (4.24 g, 12 mmol) in 100 mL of CH2Cl2 and Et3N (6 mL), and the mixture was stirred for 2 h. HCl (50 mL, 1 N) was added, and the organic layer was separated, washed with sat. NaHCO3 and brine, and dried over MgSO4. Column chromatography eluting with CH2Cl2/MeOH gave 5.20 g of compound 2 as a white solid. Yield: 95.6%; mp 105107 C; [R]23D= 85.5 (c 0.42, CHCl3). 1H NMR (300 MHz, CDCl3): δ 7.89 (s, 2H), 7.81 (d, J = 9.0 Hz, 1H), 7.28 (s, 1H), 7.22 (d, J = 8.7 Hz, 1H), 5.29 (br s, 1H), 4.84 (m, 1H), 4.61 (br s, 1H), 4.09 (s, 3H), 4.05 (s, 3H), 4.01 (s, 3H), 3.733.62 (m, 2H), 3.27 (dd, J = 16.5 Hz, J = 6.3 Hz, 1H), 3.18 (t, J = 16.5 Hz, 1H), 1.55 (s, 9H). 13 C NMR (75 MHz, CDCl3): δ 157.9, 156.2, 149.7, 148.8, 130.4, 126.7, 124.0, 123.9, 123.8, 123.4, 122.8, 115.2, 105.1, 104.2, 104.0, 80.6, 62.7, 56.2, 56.0, 55.7, 50.6, 41.0, 28.6 (3 C), 26.5. ESI-HRMS ([M + H]+) calcd for C26H32NO6 454.2230; found, 454.2246.
General Procedures for the Synthesis of 12N-Substituted12-aza-antofine (3al). The aldehyde was obtained using the same procedure as that for compound 8. Various amines were reacted with the aldehyde in MeOH, followed by the addition of HOAc and NaBH3CN, which was stirred at r.t. for 4 h. Saturated NaHCO3 was added to quench the reaction, and CH2Cl2 was used for extraction. The residue was quickly purified through a column, and TFA was used to remove the Boc group. Then TFA was removed by evaporation, and the residue was dissolved in CH2Cl2 (10 mL), to which K2CO3 (100 mg) and MgSO4 (0.5 g) were added, followed by HCHO (37%, 0.1 mL). The mixture was stirred at r.t. overnight. Chromatography gave light yellow to white solids. Yield: 40%80%. Figure 2. RT-PCR results of R-antofine, 11, and 15 at 0.01 μg/mL on major DNA replication complexes in CL15 cells at 24 and 48 h.
(S)-tert-Butyl-6,7,10-trimethoxy-3-((2-methoxy-2-oxoethylamino)methyl)-3,4-dihydrodibenzo[f,h]isoquinoline2(1H)-carboxylate (4). The Boc-aldehyde was obtained using the same procedure as that used for compound 8. Glycine methyl ester
Figure 3. Western blot analysis of R-antofine, 11, and 15 on major DNA replication complexes in CL15 cells at 24 and 48 h. 5103
dx.doi.org/10.1021/jm200330s |J. Med. Chem. 2011, 54, 5097–5107
Journal of Medicinal Chemistry
ARTICLE
Figure 4. Individual animal body weight change for control and treatment groups. hydrochloride (314 mg, 2.5 mmol) and Et3N (0.35 mL, 2.5 mmol) were added to the aldehyde in MeOH (30 mL), which was stirred for 0.5 h. Then HOAc (0.70 mL, 12 mmol) and NaBH3CN (165 mg, 2.5 mmol) were added. The mixture was stirred for 2 h until all the aldehyde disappeared. Saturated NaHCO3 was used to quench the reaction, and CH2Cl2 was used for extraction, and washed with brine, dried over MgSO4. Chromatography gave 635 mg of compound 4 as a white solid. Yield: 61% over two steps. mp 7880 C; [R]23D= 91.6 (c 0.37, CHCl3). 1H NMR (400 MHz, CDCl3): δ 7.92 (s, 1H), 7.91 (d, J = 2.4 Hz, 1H), 7.87 (brs, 1H), 7.447.42 (m, 2H), 7.29 (s, 1H), 7.257.23 (m, 1H), 5.405.29 (m, 1H), 4.924.78 (m, 1H), 4.56 (m, 1H), 4.11 (s, 3H), 4.05 (s, 3H), 4.02 (s, 3H), 3.66 (s, 3H), 3.47 3.36 (m, 2H), 3.27 (dd, J = 16.4 Hz, J = 6.4 Hz, 1H), 3.14 (d, J = 16.4 Hz, 1H), 2.81 (dd, J = 12.0 Hz, J = 8.8 Hz, 1H), 2.64 (dd, J = 12.0 Hz, J = 6.4 Hz, 1H), 1.54 (s, 9H). ESI MS m/z 525.20 (M + H)+. (S)-13N-Boc-11-oxo-13-aza-cryptopleurine (5). The ester (635 mg, 1.21 mmol) was dissolved in HCl (1.25 M in methanol, 15 mL), which was stirred at 50 C for 1 h. The solvent was removed. Thirty milliliters of MeOH and Et3N (0.3 mL) was added. The mixture was stirred at r.t. for 2 h. After normal workup, chromatography afforded 404 mg of ring-closed intermediate as a white solid. Using a procedure similar to that for compound 2, the intermediate was protected by (Boc)2O to give 456 mg of 5 as a white solid. Yield: 76% over three steps. mp 125127 C; [R]23D= 202.8 (c 0.50, CHCl3). 1H NMR (400 MHz, CDCl3): δ 7.89 (s, 1H), 7.88 (d, J = 2.8 Hz, 1H), 7.87 (d, J = 9.2 Hz, 1H), 7.23 (dd, J = 9.2 Hz, J = 2.8 Hz, 1H), 7.19 (s, 1H), 5.88 (d, J = 17.2 Hz, 1H), 4.49 (d, J = 17.2 Hz, 1H), 4.40 (d, J = 18.0 Hz, 1H), 4.10 (s, 4H), 4.05 (s, 4H), 4.01 (s, 3H), 3.873.77 (m, 2H), 3.16 (m, 2H), 1.51 (s, 9H). ESI MS m/z 493.10 (M + H)+, 985.20 (2M + H)+.
(S)-13N-Boc-13-aza-cryptopleurine (6). To a solution of the amide (456 mg, 0.93 mmol) in THF (30 mL) was added BMS (2 M in THF, 2.79 mL), which was stirred at r.t. overnight. Five milliliters of MeOH was added, and the mixture was refluxed for 1 h. Chromatography afforded 400 mg of 6 as a white solid. Yield: 90%. mp 116118 C; [R]23D= 182.5 (c 0.28, CHCl3). 1H NMR (400 MHz, CDCl3): δ 7.887.87 (m, 2H), 7.76 (d, J = 8.8 Hz, 1H), 7.20 (s, 1H), 7.19 (dd, J = 8.8 Hz, J = 2.8 Hz, 1H), 4.44 (d, J = 15.2 Hz, 1H), 4.29 (m, 1H), 4.11 (m, 1H), 4.09 (s, 3H), 4.05 (s, 3H), 4.00 (s, 3H), 3.66 (d, J = 15.2 Hz, 1H), 3.15 (m, 2H), 3.04 (dd, J = 16.4 Hz, J = 3.2 Hz, 1H), 2.87 (m, 1H), 2.80 (dd, J = 16.4 Hz, J = 11.8 Hz, 1H), 2.572.52 (m, 1H), 2.45 (dt, J = 12.0 Hz, 3.2 Hz, 1H). ESI MS m/z 479.10 (M + H)+. General Procedures for the Synthesis of 13N-Substituted13-aza-cryptopleurine (7aq). (a) 13N-Boc-13-aza-cryptopleurine 6 was stirred in HCl/MeOH for 2 h before the solvent was removed in vacuo. The solid was collected and washed sequentially with cold MeOH and ether. Then the solid was dissolved in 10 mL of anhydrous CH2Cl2 and Et3N (0.1 mL), to which RCOCl was added at 0 C. The mixture was stirred at r.t. for 4 h before 1 N HCl was added. After routine workup, chromatography afforded from light orange to white solids. Yield: 70%80%. (b) The same procedures (reductive amination) as those for compound 4 were used. Yield: 50%- 80%.
(S,E/Z)-6,7,10-Trimethoxy-3-(2-methoxyvinyl)-1,2,3,4-tetrahydrodibenzo[f,h]isoquinoline (8). N-Boc-alcohol 2 (1.812 g, 4 mmol) was dissolved in CH2Cl2 (50 mL) and Et3N (1.95 mL, 14 mmol) at 0 C, to which Py 3 SO3 (2.23 g, 14 mmol) in 10 mL of DMSO was added dropwise. The ice bath was then removed, and the mixture was stirred at r.t. until 2 disappeared by TLC. HCl (1 N) was added, and the 5104
dx.doi.org/10.1021/jm200330s |J. Med. Chem. 2011, 54, 5097–5107
Journal of Medicinal Chemistry
ARTICLE
Figure 5. In vivo antitumor activity of compound 11.
Figure 6. Comparison of compound 11 and R-cryptopleurine against human umbilical vein endothelial cells (HUVECs). organic layer was then separated and washed with sat. NaHCO3, brine, and dried over MgSO4. In another flask, Ph3P+CH2OMeCl (2.74 g, 8 mmol) was added to KOtBu (875 mg, 7.8 mmol) at 0 C under N2,
which was stirred for 0.5 h. The aldehyde in 20 mL of CH2Cl2 was added dropwise, followed by the removal of the ice bath. The reaction was kept for 23 h before sat. NH4Cl was poured into the mixture. CH2Cl2 was 5105
dx.doi.org/10.1021/jm200330s |J. Med. Chem. 2011, 54, 5097–5107
Journal of Medicinal Chemistry used for extraction, and the organic layers were washed with sat. NaHCO3 and brine, dried over MgSO4. Chromatography afforded 1.5 g of compound 8 as a light yellow solid. Yield: 78% (Z/E = 1/2.5) for two steps. mp 150152 C; [R]23D= 108.1 (c 0.59, CHCl3). 1H NMR (400 MHz, CDCl3, E/Z = 2.5:1): δ 7.947.91 (m, 2H), 7.88 (d, J = 9.2 Hz, 1H), 7.29 (s, 1H), 7.267.22 (m, 1H), 6.30 (d, J = 12.8 Hz, 0.68H, E isomer), 5.86 (dd, J = 6.4 Hz, J = 1.6 Hz, 0.28 Hz, Z isomer), 5.72 (m, 0.31H, Z isomer), 5.305.25 (d, J = 16.8 Hz, 1H+0.67H, E isomer), 4.80 (dd, J = 12.8 Hz, J = 8.4 Hz, 0.71 H, E isomer), 4.59 (d, J = 17.2 Hz, 1H), 4.42 (dd, J = 8.0 Hz, J = 6.4 Hz, 0.29H, Z isomer), 4.124.11 (m, 3H), 4.054.04 (m, 3H), 4.034.02 (m, 3H), 3.62 (s, 0.71H, Z isomer), 3.443.32 (m, 1H), 3.36 (s, 2H, E isomer), 3.13 (d, J = 16.0 Hz, 1H), 1.55 (s, 9H). ESI MS m/z 480.05 (M + H)+.
(S)-2-(6,7,10-Trimethoxy-1,2,3,4-tetrahydrodibenzo[f,h]isoquinolin-3-yl)acetaldehyde (9). Compound 8 (1.5 g, 3.12 mmol) was dissolved in THF (50 mL) and H2O (5 mL), to which Hg(OAc)2 (3.0 g, 9.36 mmol) was added at 0 C. Then the ice bath was removed, and the mixture was stirred overnight. Freshly prepared sat. KI (50 mL) was added dropwise at 0 C for 10 min, and CH2Cl2 was used for extraction. The organic layers were collected and washed with brine, dried over MgSO4. Chromatography furnished 1.09 g of compound 9 as a light yellow foam. Yield: 75%. mp 98100 C; [R]23D= 94.4 (c 0.68, CHCl3). 1H NMR (400 MHz, CDCl3): δ 9.79 (s, 1H), 7.93 (s, 1H), 7.91 (d, J = 2.4 Hz, 1H), 7.85 (brs, 1H), 7.267.24 (m, 2H), 5.385.29 (m, 2H), 4.58 (d, J = 16.0 Hz, 1H), 4.11 (s, 3H), 4.05 (s, 3H), 4.02 (s, 3H), 3.38 (dd, J = 16.0 Hz, J = 6.4 Hz, 1H), 3.12 (d, J = 16.4 Hz, 1H), 2.702.59 (m, 2H), 1.53 (s, 9H). ESI MS m/z 466.10 (M + H)+, 488.15 (M + Na)+.
General Procedures for the Synthesis of 12N-Substituted12-aza-cryptopleurine (10aj). Similar procedures as those used for compound 3al were used. Yield: 50%70%.
(S)-13-Oxa-cryptopleurine (11) and (R)-13-Oxa-cryptopleurine (12). Compound 2 (177 mg, 0.5 mmol) was suspended in
dry CH2Cl2 (15 mL) and Et3N (0.14 mL, 1.00 mmol) at 0 C, to which chloroacetyl chloride (40 μL, 0.50 mmol) in 1 mL of CH2Cl2 was slowly added dropwise. The mixture was stirred at 0 C for 5 h before 1 N HCl was added. The organic layer was separated and washed with sat. NaHCO3 and brine, dried over MgSO4. CH2Cl2 was removed, and the residue was dissolved in anhydrous THF (5 mL), to which NaH (2.1 equiv) was added at r.t. followed by reflux for 2 h. Saturated NH4Cl was added, and CH2Cl2 was used for extraction. After workup, the organic layer was dried over MgSO4. Then the residue was dissolved in 10 mL of anhydrous THF, and BMS (1.50 mL, 6 mmol) was added, which was stirred at r.t. overnight. MeOH (5 mL) was added, and the mixture was refluxed for 1 h. Chromatography gave 73 mg of 11 as a white solid. Yield: 38.5% for three steps; white solid; mp 199201 C; [R]23D= 134.6 (c 0.52, CHCl3). 1H NMR (300 MHz, CDCl3): δ 7.867.85 (m, 2H), 7.74 (d, J = 9.0 Hz, 1H), 7.18 (dd, J = 9.0 Hz, J = 2.4 Hz, 1H), 7.14 (s, 1H), 4.39 (d, J = 15.6 Hz, 1H), 4.12 (m, 2H), 4.08 (s, 3H), 4.03 (s, 3H), 4.00 (s, 3H), 3.913.83 (m, 1H), 3.66 (d, J = 15.6 Hz, 1H), 3.51 (dd, J = 11.1 Hz, J = 9.0 Hz, 1H), 3.06 (d, J = 11.7 Hz, 1H), 2.912.87 (m, 1H), 2.762.59 (m, 3H). ESI MS m/z 380.05 (M + H)+. For 12: mp 196198 C; [R]23D= 139.6 (c 0.26, CHCl3). 1H NMR (300 MHz, CDCl3): δ 7.907.89 (m, 2H), 7.78 (d, J = 9.0 Hz, 1H), 7.20 (dd, J = 9.0 Hz, J = 2.7 Hz, 1H), 7.20 (s, 1H), 4.44 (d, J = 15.6 Hz, 1H), 4.174.08 (m, 1H), 4.10 (s, 3H), 4.05 (s, 3H), 4.01 (s, 3H), 4.044.00 (m, 1H), 3.923.84 (m, 1H), 3.72 (d, J = 15.3 Hz, 1H), 3.54 (dd, J = 11.1 Hz, J = 9.0 Hz, 1H), 3.09 (d, J = 11.4 Hz, 1H), 2.982.94 (m, 1H), 2.772.61 (m, 3H). 13C NMR (75 MHz, CDCl3): δ 157.5, 149.4, 148.4, 130.2, 126.4, 125.1, 124.1, 123.6, 123.4, 123.3, 115.0, 104.7, 103.8, 103.6, 72.3, 67.4, 56.1, 56.0, 55.9, 55.5, 55.1, 54.4, 28.5. ESI MS m/z 380.05 (M + H)+.
(S)-12-Oxa-cryptopleurine (13) and (R)-12-Oxa-cryptopleurine (14). Aldehyde 9 (100 mg, 0.21 mmol) was dissolved in MeOH, to which NaBH4 (19 mg, 0.50 mmol) was added in one portion. The mixture was stirred for 1 h, and sat. NaHCO3 was added. After normal
ARTICLE
workup, 80 mg of white solid was obtained, which was used without further purification. The residue was dissolved in CH2Cl2 (10 mL), to which MgSO4, K2CO3, and HCHO were sequentially added. The mixture was stirred overnight. After workup, chromatography gave 32 mg of 13 as a white solid. Yield: 40% for two steps; light yellow solid; mp 195197 C; [R]23D= 56.8 (c 0.60, CHCl3). 1H NMR (300 MHz, CDCl3): δ 7.897.88 (m, 2H), 7.75 (d, J = 9.3 Hz, 1H), 7.22 (s, 1H), 7.19 (dd, J = 9.0 Hz, J = 2.7 Hz, 1H), 4.79 (d, J = 8.4 Hz, 1H), 4.41 (d, J = 15.3 Hz, 1H), 4.19 (dd, J = 11.1 Hz, J = 5.1 Hz, 1H), 4.10 (s, 3H), 4.05 (s, 3H), 4.03 (m, 1H), 4.01 (s, 3H), 3.753.65 (m, 2H), 3.17 (dd, J = 15.9 Hz, J = 3.6 Hz, 1H), 2.952.80 (m, 2H), 2.061.92 (m, 1H), 1.74 (m, 1H); ESI MS m/z 380.05 (M + H)+. For 14: mp 191192 C; [R]23D= 51.2 (c 0.60, CHCl3). 1H NMR (300 MHz, CDCl3): δ 7.877.86 (m, 2H), 7.73 (d, J = 9.0 Hz, 1H), 7.20 (s, 1H), 7.18 (dd, J = 9.6 Hz, J = 3.0 Hz, 1H), 4.77 (d, J = 8.1 Hz, 1H), 4.38 (d, J = 15.3 Hz, 1H), 4.18 (dd, J = 11.1 Hz, J = 4.8 Hz, 1H), 4.09 (s, 3H), 4.04 (s, 3H), 3.99 (s, 3H), 3.98 (d, J = 8.1 Hz, 1H), 3.733.61 (m, 2H), 3.13 (dd, J = 15.9 Hz, J = 3.6 Hz, 1H), 2.912.72 (m, 2H), 2.021.90 (m, 1H), 1.721.67 (m, 1H). 13C NMR (75 MHz, CDCl3): δ 157.7, 149.6, 148.5, 130.3, 126.6, 124.7, 124.2, 123.8, 123.7, 123.6, 115.0, 104.9, 104.5, 103.9, 86.5, 68.1, 56.1, 56.0, 55.7, 55.4, 48.0, 33.5, 31.3. ESI MS m/z 380.05 (M + H)+. Compound 16. Aldehyde 9 (140 mg, 0.30 mmol) was dissolved in MeOH, to which NaBH4 (76 mg, 2 mmol) was added. The resulting mixture was stirred at r.t. for 1 h before sat. NaHCO3 was added. The mixture was extracted with CH2Cl2, and the organic layers were combined, washed with brine, and dried over Na2SO4. Then the solvent was removed under reduced pressure and redissolved in TFA/CH2Cl2 (1:1, 10 mL), which was stirred for 0.5 h. The mixture was concentrated to remove TFA. The residue was subject to treatment with chloroacetyl chloride, and the rest of the synthesis was similar to that of compound 11. After chromatography, 37 mg of 16 was obtained as a light yellow solid in 24% yield over five steps. mp 183185 C; [R]23D= 114.7 (c 0.19, CHCl3). 1H NMR (400 MHz, CDCl3): δ 7.90 (s, 1H), 7.88 (d, J = 2.4 Hz, 1H), 7.75 (d, J = 9.2 Hz, 1H), 7.24 (s, 1H), 7.19 (dd, J = 9.2 Hz, J = 2.4 Hz, 1H), 4.43 (d, J = 15.2 Hz, 1H), 4.10 (s, 3H), 4.05 (m, 4H), 4.01 (m, 4H), 3.983.90 (m, 3H), 3.203.13 (m, 1H), 3.083.01 (m, 3H), 2.962.91 (m, 1H), 2.252.19 (m, 1H), 2.172.11 (m, 1H). 13C NMR (100 MHz, CDCl3): δ 157.7, 149.6, 148.6, 130.3, 126.9, 126.6, 125.4, 124.2, 123.9, 123.7, 115.1, 104.9, 104.1, 104.0, 69.9, 66.5, 59.0, 57.6, 57.0, 56.2, 56.1, 55.7, 36.7, 35.5. ESI MS m/z 394.10 (M + H)+.
’ ASSOCIATED CONTENT
bS
Supporting Information. Compound data for 3al, 7aq, and 10aj, biological studies, and HPLC analysis of final compounds. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*(Q.S.) Phone: 919-843-6325. Fax: 919-966-3893. E-mail:
[email protected]. (P.-C.Y.) Phone: 886-2-2356-2185. Fax: 886-2-2322-4793. E-mail:
[email protected]. (K.-H.L.) Phone: 919-962-0066. Fax: 919-966-3893. E-mail:
[email protected].
’ ACKNOWLEDGMENT This study was supported by grant CA17625-32 from the National Cancer Institute awarded to K.H.L. and grant DOH98-TD-G111-007 from the National Research Program for Genomic Medicine awarded to P.C.Y. This study was also supported in part by the Department of Health Cancer Research Center of Excellence (DOH-100-TD-C-111-05). 5106
dx.doi.org/10.1021/jm200330s |J. Med. Chem. 2011, 54, 5097–5107
Journal of Medicinal Chemistry
’ ABBREVIATIONS USED MOA, mechanism of action; NCI, National Cancer Institute; HIF-1, hypoxia-inducible factor 1; TS, thymidylate synthase; DHFR, dihydrofolate reductase; AP-1, activator protein-1; CRE, cAMP response element; CDK, cyclin-dependent kinase; CNS, central neural system; SAR, structureactivity relationship; BBB, bloodbrain barrier; PSA, polar surface area; ORC, origin recognition complex; CDC6, cell division control protein 6; RTPCR, reverse transcription polymerase chain reaction; HUVECs, human umbilical vein endothelial cells ’ REFERENCES (1) Gellert, E. The indolizidine alkaloids. J. Nat. Prod. 1982, 45 (1), 50–73. (2) Xi, Z.; Zhang, R.; Yu, Z.; Ouyang, D. The interaction between tylophorine B and TMV RNA. Bioorg. Med. Chem. Lett. 2006, 16 (16), 4300–4304. (3) Wang, K.; Su, B.; Wang, Z.; Wu, M.; Li, Z.; Hu, Y.; Fan, Z.; Mi, N.; Wang, Q., Synthesis and antiviral activities of phenanthroindolizidine alkaloids and their derivatives. J. Agric. Food Chem. 2010, 58 (5), 2703-2709. (4) Baumgartner, B.; Erdelmeier, C. A. J.; Wright, A. D.; Rali, T.; Sticher, O. An antimicrobial alkaloid from Ficus septica. Phytochemistry 1990, 29 (10), 3327–3330. (5) Bhutani, K. K.; Sharma, G. L.; Ali, M. Plant based antiamoebic drugs; Part I. Antiamoebic activity of phenanthroindolizidine alkaloids; common structural determinants of activity with emetine. Planta Med. 1987, 53 (6), 532–536. (6) Banwell, M. G.; Bezos, A.; Burns, C.; Kruszelnicki, I.; Parish, C. R.; Su, S.; Sydnes, M. O. C8c-C15 monoseco-analogues of the phenanthroquinolizidine alkaloids julandine and cryptopleurine exhibiting potent anti-angiogenic properties. Bioorg. Med. Chem. Lett. 2006, 16 (1), 181–185. (7) Yang, C.-W.; Chen, W.-L.; Wu, P.-L.; Tseng, H.-Y.; Lee, S.-J. Anti-inflammatory mechanisms of phenanthroindolizidine alkaloids. Mol. Pharmacol. 2006, 69 (3), 749–758. (8) Yang, C.-W.; Chuang, T.-H.; Wu, P.-L.; Huang, W.-H.; Lee, S.-J. Anti-inflammatory effects of 7-methoxycryptopleurine and structureactivity relations of phenanthroindolizidines and phenanthroquinolizidines. Biochem. Biophys. Res. Commun. 2007, 354 (4), 942–948. (9) NCI 60-cell assay results can be found at http://dtp.nci.nih.gov/ dtpstandard/dwindex/index.jsp. (10) Gao, W.; Lam, W.; Zhong, S.; Kaczmarek, C.; Baker David, C.; Cheng, Y.-C. Novel mode of action of tylophorine analogs as antitumor compounds. Cancer Res. 2004, 64 (2), 678–688. (11) Li, Z.; Jin, Z.; Huang, R. Isolation, total synthesis and biological activity of phenanthroindolizidine and phenanthroquinolizidine alkaloids. Synthesis 2001, 16, 2365–2378. (12) Chemler, S. R. Phenanthroindolizidines and phenanthroquinolizidines: promising alkaloids for anti-cancer therapy. Curr. Bioact. Compd. 2009, 5 (1), 2–19. (13) Cusack, J. C., Jr.; Liu, R.; Baldwin, A. S., Jr. Inducible chemoresistance to 7-ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxycamptothe cin (CPT-11) in colorectal cancer cells and a xenograft model is overcome by inhibition of nuclear factor-kappaB activation. Cancer Res. 2000, 60 (9), 2323–2330. (14) Huang, M. T.; Grollman, A. P. Mode of action of tylocrebrine effects on protein and nucleic-acid synthesis. Mol. Pharmacol. 1972, 8 (5), 538–550. (15) Gupta, R. S.; Siminovitch, L. Mutants of CHO cells resistant to the protein synthesis inhibitors, cryptopleurine and tylocrebrine: genetic and biochemical evidence for common site of action of emetine, cryptopleurine, tylocrebrine, and tubulosine. Biochemistry 1977, 16 (14), 3209–3214. (16) Gupta, R. S.; Krepinsky, J. J.; Siminovitch, L. Structural determinants responsible for the biological activity of (-)-emetine,
ARTICLE
(-)-cryptopleurine, and (-)-tylocrebrine: structure-activity relationship among related compounds. Mol. Pharmacol. 1980, 18 (1), 136–143. (17) Dolz, H.; Vazquez, D.; Jimenez, A. Quantitation of the specific interaction of [14a-3H]cryptopleurine with 80S and 40S ribosomal species from the yeast Saccharomyces cerevisiae. Biochemistry 1982, 21 (13), 3181–3187. (18) Cai, X. F.; Jin, X.; Lee, D.; Yang, Y. T.; Lee, K.; Hong, Y.-S.; Lee, J.-H.; Lee, J. J. Phenanthroquinolizidine alkaloids from the roots of Boehmeria pannosa potently inhibit hypoxia-inducible factor-1 in AGS human gastric cancer cells. J. Nat. Prod. 2006, 69 (7), 1095–1097. (19) Rao, K. N.; Bhattacharya, R. K.; Venkatachalam, S. R. Inhibition of thymidylate synthase and cell growth by the phenanthroindolizidine alkaloids pergularinine and tylophorinidine. Chem.-Biol. Interact. 1997, 106 (3), 201–212. (20) Rao, K. N.; Bhattacharya, R. K.; Veankatachalam, S. R. Inhibition of thymidylate synthase by pergularinine, tylophorinidine and deoxytubulosine. Indian J. Biochem. Biophys. 1999, 36 (6), 442–448. (21) Rao, K. N.; Venkatachalam, S. R. Inhibition of dihydrofolate reductase and cell growth activity by the phenanthroindolizidine alkaloids pergularinine and tylophorinidine: the in vitro cytotoxicity of these plant alkaloids and their potential as antimicrobial and anticancer agents. Toxicol. in Vitro 2000, 14 (1), 53–59. (22) Gao, W.; Chen, A. P.-C.; Leung, C.-H.; Gullen, E. A.; Fuerstner, A.; Shi, Q.; Wei, L.; Lee, K.-H.; Cheng, Y.-C. Structural analogs of tylophora alkaloids may not be functional analogs. Bioorg. Med. Chem. Lett. 2008, 18 (2), 704–709. (23) Suffness, M.; Douros, J. Anticancer Agents Based on Natural Product Models; Academic Press: New York, 1980; pp 465487. (24) Gao, W.; Bussom, S.; Grill, S. P.; Gullen, E. A.; Hu, Y.-C.; Huang, X.; Zhong, S.; Kaczmarek, C.; Gutierrez, J.; Francis, S.; Baker, D. C.; Yu, S.; Cheng, Y.-C. Structure-activity studies of phenanthroindolizidine alkaloids as potential antitumor agents. Bioorg. Med. Chem. Lett. 2007, 17 (15), 4338–4342. (25) Wei, L.; Shi, Q.; Bastow Kenneth, F.; Brossi, A.; MorrisNatschke Susan, L.; Nakagawa-Goto, K.; Wu, T.-S.; Pan, S.-L.; Teng, C.-M.; Lee, K.-H. Antitumor agents 253. Design, synthesis, and antitumor evaluation of novel 9-substituted phenanthrene-based tylophorine derivatives as potential anticancer agents. J. Med. Chem. 2007, 50 (15), 3674–3680. (26) Lin, J. C.; Yang, S. C.; Hong, T. M.; Yu, S. L.; Shi, Q.; Wei, L.; Chen, H. Y.; Yang, P. C.; Lee, K. H. Phenanthrene-based tylophorine-1 (PBT-1) inhibits lung cancer cell growth through the Akt and NFkappaB pathways. J. Med. Chem. 2009, 52 (7), 1903–1911. (27) Yang, X.; Shi, Q.; Liu, Y. N.; Zhao, G.; Bastow, K. F.; Lin, J. C.; Yang, S. C.; Yang, P. C.; Lee, K. H. Antitumor agents 268. Design, synthesis, and mechanistic studies of new 9-substituted phenanthrenebased tylophorine analogues as potent cytotoxic agents. J. Med. Chem. 2009, 52 (16), 5262–5268. (28) Wang, K.; Wang, W.; Wang, Q.; Huang, R. Efficient synthesis of aza-phenanthroindolizidine and aza-phenanthroquinolizidine and anticancer activities. Lett. Org. Chem. 2008, 5 (5), 383–390. (29) Yang, X. M.; Shi, Q.; Bastow, K. F.; Lee, K. H. Antitumor Agents. 274. A new synthetic strategy for E-ring SAR study of antofine and cryptopleurine analogues. Org. Lett. 2010, 12 (7), 1416–1419. (30) Alavijeh, M. S.; Chishty, M.; Qaiser, M. Z.; Palmer, A. M. Drug metabolism and pharmacokinetics, the blood-brain barrier, and central nervous system drug discovery. NeuroRx 2005, 2 (4), 554–571. (31) Clark, D. E. In silico prediction of blood-brain barrier permeation. Drug Discovery Today 2003, 8 (20), 927–933. (32) van de Waterbeemd, H.; Camenisch, G.; Folkers, G.; Chretien, J. R.; Raevsky, O. A. Estimation of blood-brain barrier crossing of drugs using molecular size and shape, and H-bonding descriptors. J. Drug Target 1998, 6 (2), 151–165. (33) http://preadmet.bmdrc.org (34) Wang, Y.; Gao, W.; Svitkin, Y. V.; Chen, A. P.; Cheng, Y. C. DCB-3503, a tylophorine analog, inhibits protein synthesis through a novel mechanism. PLoS One 2010, 5 (7), e11607. 5107
dx.doi.org/10.1021/jm200330s |J. Med. Chem. 2011, 54, 5097–5107
